Keysson Vieira Fernandes

Identification of Critical Amino Acids in the IgE Epitopes of Ric c 1 and Ric c 3 and the Application of Glutamic Acid as an IgE Blocker

Background The allergenicity of Ricinus communis L. (castor bean, Euphorbiaceae) is associated with components of its seeds and pollen. Castor bean allergy has been described not only in laboratory workers, but also in personnel working in oil processing mills, fertilizer retail, the upholstery industry and other industrial fields. In the present study, we describe the critical amino acids in the IgE-binding epitopes in Ric c 1 and Ric c 3, two major allergens of R. communis. In addition, we also investigate the cross-reactivity between castor bean and some air and food allergen extracts commonly used in allergy diagnosis. Methodology/Principal Findings The IgE reactivity of human sera from atopic patients was screened by immune-dot blot against castor bean allergens. Allergenic activity was evaluated in vitro using a rat mast cell activation assay and by ELISA. Cross-reactivity was observed between castor bean allergens and extracts from shrimp, fish, gluten, wheat, soybean, peanut, corn, house dust, tobacco and airborne fungal allergens. We observed that treatment of rat and human sera (from atopic patients) with glutamic acid reduced the IgE-epitope interaction. Conclusions/Significance The identification of glutamic acid residues with critical roles in IgE-binding to Ric c 3 and Ric c 1 support the potential use of free amino acids in allergy treatment.

Simultaneous allergen inactivation and detoxification of castor bean cake by treatment with calcium compounds

Ricinus communis L. is of great economic importance due to the oil extracted from its seeds. Castor oil has been used for pharmaceutical and industrial applications, as a lubricant or coating agent, as a component of plastic products, as a fungicide or in the synthesis of biodiesel fuels. After oil extraction, a castor cake with a large amount of protein is obtained. However, this by-product cannot be used as animal feed due to the presence of toxic (ricin) and allergenic (2S albumin) proteins. Here, we propose two processes for detoxification and allergen inactivation of the castor cake. In addition, we establish a biological test to detect ricin and validate these detoxification processes. In this test, Vero cells were treated with ricin, and cell death was assessed by cell counting and measurement of lactate dehydrogenase activity. The limit of detection of the Vero cell assay was 10 ng/mL using a concentration of 1.6 × 105 cells/well. Solid-state fermentation (SSF) and treatment with calcium compounds were used as cake detoxification processes. For SSF, Aspergillus niger was grown using a castor cake as a substrate, and this cake was analyzed after 24, 48, 72, and 96 h of SSF. Ricin was eliminated after 24 h of SSF treatment. The cake was treated with 4 or 8% Ca(OH)2 or CaO, and both the toxicity and the allergenic properties were entirely abolished. A by-product free of toxicity and allergens was obtained.

Activity of recombinant and natural defensins from Vigna unguiculata seeds against Leishmania amazonensis.

Antimicrobial peptides (AMPs), which are differentiated from other antibiotic peptides, such as gramicidins and polymyxins, because they are synthesized by large enzymatic complex and bear modified amino acids including d-amino acids, are short polymers of l-amino acids synthesized by ribosomes upon which all living organisms rely to defend themselves from invaders or competitor microorganisms. AMPs have received a great deal of attention from the scientific community as potential new drugs for neglected diseases such as Leishmaniasis. In plants, they include several families of compounds, including the plant defensins. The aim of the present study was to improve the expression of recombinant defensin from Vigna unguiculata seeds (Vu-Defr) and to test its activity against Leishmania amazonensis promatigotes. Recombinant expression was performed in LB and TB media and under different conditions. The purification of Vu-Defr was achieved by immobilized metal ion affinity and reversed-phase chromatography. The purified Vu-Defr was analyzed by circular dichroism (CD), and its biological activity was tested against L. amazonenis promastigotes. To demonstrate that the recombinant production of Vu-Defr did not interfere with its fold and biological activity, the results of all experiments were compared with the results from the natural defensin (Vu-Def). The CD spectra of both peptides presented good superimposition indicating that both peptides present very similar secondary structure and that the Vu-Defr was correctly folded. L. amazonensis treated with Vu-Defr led to the elimination of 54.3% and 46.9% of the parasites at 24 and 48h of incubation time, respectively. Vu-Def eliminated 50% and 54.8% of the parasites at 24 and 48 h, respectively. Both were used at a concentration of 100 μg/mL. These results suggested the potential for plant defensins to be used as new antiparasitic substances.

Bioprocess development for biolubricant production using microbial oil derived via fermentation from confectionery industry wastes

Microbial oil produced from confectionery and wheat milling side streams has been evaluated as novel feedstock for biolubricant production. Nutrient-rich fermentation media were produced by a two-step bioprocess involving crude enzyme production by solid state fermentation followed by enzymatic hydrolysis of confectionery industry waste. Among 5 yeast strains and 2 fungal strains cultivated on the crude hydrolysate, Rhodosporidium toruloides and Cryptococcus curvatus were selected for further evaluation for biolubricant production based on fermentation efficiency and fatty acid composition. The extracted microbial oils were enzymatically hydrolysed and the free fatty acids were esterified by Lipomod 34-MDP in a solvent-free system with trimethylolpropane (TMP) and neopentyl glycol (NPG). The highest conversion yields were 88% and 82.7% for NPG esters of R. toruloides and C. curvatus, respectively. This study also demonstrates that NPG esters produced from microbial oil have promising physicochemical properties for bio-based lubricant formulations that could substitute for conventional lubricants.

Approaches for the Detection of Toxic Compounds in Castor and Physic Nut Seeds and Cakes

The worldwide search for new fuel sources has grown during the last decades due to two main factors: the global concern about environmental issues and the high price of petrole‐ um. Biodiesel is a type of biofuel that is already used in many countries, and its usage will most likely increase over the next few years. Biodiesel can be produced using different tech‐ nologies and raw materials, such as vegetable oils, animal fats and microalgae oil. However, despite the wide range of oil sources for biodiesel production, vegetable oils are primarily used for this purpose. The choice of oilseed to be planted for biodiesel production depends on many factors, including the regional climate and soil conditions. The biodiesel industries in the US primarily use soybean oil, whereas in Europe, rapeseed is primarily used for bio‐ diesel production. In tropical countries, biodiesel is produced from plants that grow in these tropical areas, such as palm, physic nut and castor bean.